Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays

Abstract

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.

Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

“…Protocols for nanogram-scale verification have been constructed for over twenty kinds of glycoprotein marker candidates toward the forthcoming validation phase 2 , where in turn an immune-based sandwich assay should work better, i.e. lectinoverlay antibody microarray (20). For realization of this, selection of appropriate lectin probes is critical to differentiate disease-specific glycan alterations.…”

“…However, taking into consideration the critical glycosylation changes occurring on diverse glycoproteins, differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen et al (20) have recently developed a lectin-overlay antibody microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated assays with more than 30 lectins to search for critical differences in glycan structures.…”

Advances in purification and separation of posttranslationally modified proteins

“…An alternative antibody-lectin sandwich version captures specific glycoproteins by antibodies and hybridized arrays are then incubated with fluorescently or biotin-labeled lectins for detection. This approach is primarily used for detecting and comparing differences in glycan structures, but MS can be used to identify the glycoproteins within a sample [177][178][179]. Glycoproteins may also be detected by mobility shifts, in either the pI or MW dimension, in diagonal separations after enzymatic or chemical (e.g.…”

Modification of Glycan Expression in Staphylococcus epidermidis to Prevent Biofilm Formation

“…Altered glycan molecules have been reported as indicators for the development of malignant cells (Rosenfeld et al, 2007); and can be applied as biomarkers for diagnosis of infections (Chen et al, 2007).…”